Diabetes affects 9% of the United States population and approximately one-third of these patients experience chronic neuropathic pain, commonly referred to as painful diabetic neuropathy (PDN). PDN is difficult to manage as analgesic treatments are only effective in a small subset of PDN patients. The development of analgesics for pain associated with diabetes, in particular type 2 diabetes, is stalled by our incomplete understanding of the underlying mechanisms of PDN. An important new clue comes from the recent finding that methylglyoxal (MG), a highly reactive dicarbonyl product of glycolysis that accumulates with hyperglycemia, is particularly high in patients with PDN. MG causes non-enzymatic glycation of proteins. The resulting protein adducts, or advanced glycation end products (MG-AGEs), are toxic and contribute to diabetic complications including PDN. Our central hypothesis is that elevated MG in type 2 diabetes causes pain and that this can be alleviated with new classes of drugs targeting MG itself, TRPA1, AC1, Epac, and PPAR?. To test the hypothesis that MG drives neuropathic pain (PDN) in type 2 diabetes, Specific Aim 1 will first determine whether elevations in MG and its metabolizing enzyme, Glyoxalase-1, occur in pain processing tissues in the hereditary Leprdb/db (db/db) mouse and Zucker Diabetic Fatty (ZDF) rat models of type 2 diabetes. We then ask whether a promising new class of MG-scavenging peptides will alleviate affective pain and spinal pain transmission. Our preliminary data indicate that genetic deletion or pharmacological inhibition of TRPA1, a glycation target of MG, blocks MG-induced pain. Indeed, TRPA1 is a leading target for the development of new analgesics for chronic pain, but has not been tested in models of type 2 PDN. To fill this gap, Specific Aim 2 will test the hypothesis that TRPA1 antagonists reduce affective pain and spinal pain transmission in db/db mice and ZDF rats. Consequent to TRPA1 channel opening (e.g. by MG), the resulting Ca2+ influx into the cell leads to the activation of Ca2+-sensitive proteins, which includes adenylyl cyclase I (AC1). Our data indicate that selective inhibition of AC1 with NB001 blocks type 2 PDN. AC1 generates the intracellular second messenger, cAMP, which targets not only protein kinase A but also exchange protein directly activated by cAMP (Epac). Specific Aim 3 will use novel Epac1 and Epac2 small molecule inhibitors to determine which of these targets drive PDN. Among the 33 original research articles, reviews, and a book published during the previous funding cycle (22 with the PI as first or senior author), our Progress includes the discovery that pioglitazone, a peroxisome proliferator-activated receptor gamma (PPAR?) agonist that is FDA- approved to treat diabetes, acts at dorsal horn neurons to inhibit the chronic pain associated with cutaneous inflammation and traumatic nerve injury. Our new data indicate additional efficacy in PDN and MG-induced pain, with surprisingly robust analgesic effects in females. Specific Aim 4 proposes to study PPAR? mechanism of action in db/db and ZDF, with a new focus on sex differences in spinal nociceptive transmission.